A single nucleotide polymorphism (SNP), which is a genetic variation caused by substitution of one nucleotide in DNA nucleotides, brings individual difference for each person on a cause of disease, reaction to a therapeutic agent, and the like. Attention has been focused on detection and confirmation of the SNP since they are connected to new drug development as well as personalized medicine.
In order to rapidly detect the SNP, various detecting methods in which a real-time PCR technology is applied have been used. As a representative example of the various detecting methods, there are an analysis method using DNA intercalating fluorescent material, a method of using a DNA probe, a method of using a PNA probe, and the like.
A single nucleotide polymorphism analysis method using DNA intercalating fluorescent material may differentiate a change in nucleotide of PCR amplicon by a saturation concentration of the DNA intercalating fluorescent material without additional operation with respect to a product obtained after performing a PCR reaction. However, the method has a limitation in using the DNA intercalating fluorescent material, and has disadvantages in that the only one single nucleotide is capable of being analyzed and a program for analyzing a melting curve should be used [Kirk M. Ririe, et al., ANALYTICAL BIOCHEMISTRY 245: 154160, 1997; U. Hladnik et al., Clin Exp Med 2:105108, 2002].
As the DNA probe used for the melting curve analysis, there are an MGB Taqman probe, a Molecular Beacon (MB) probe, and a binary probe. The above-listed methods have an advantage in that an excellent differentiable ability of the SNP is shown, but have a disadvantage in that it is difficult to detect the adjacent SNPs by one reaction because the minimum length of the DNA probe is long to be about 20-40 mer [Hidefumi Sasaki et al., Clin Cancer Res 11:2924-2929, 2005; Manna Zhang et al., HEPATOLOGY 36:3, 2002].
A melting curve analysis method using a PNA probe characterized by containing negative charge molecules has advantages in that the PNA probe is capable of rapidly and strongly coupling to a target DNA as compared to the existing PNA probe, and due to the strong coupling ability, a probe having a short length is capable of being used, such that adjacent single nucleotide polymorphism is capable of being detected.